Contribution of serum protein association to discrepancy between the in vivo and in vitro UDS results for 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843).
U-89843 has been shown to undergo biotransformation, both in vitro and in vivo, to form U-97924 as a major primary metabolite. U-89843 was found to be positive in an in vitro UDS mutagenesis screen conducted with primary rat hepatocytes in serum-free media. In contrast to in vitro results, no evidence of genetic toxicity of U-89843 was observed in rats in the in vivo/in vitro version of the UDS test with single oral doses up to 1400 mg/kg. The negative results may be related to more robust in vivo detoxification mechanisms or relatively lower exposure to reactive metabolites formed by bioactivation of U-89843 as compared to that observed in the serum-free in vitro hepatocyte test system. Further studies showed rat serum suppressed the in vitro metabolism of U-89843 as well as the formation of the corresponding hydroxylated metabolite, U-97924, the putative precursor of proposed reactive electrophilic metabolite. The measured in vivo systemic clearance of U-89843 (0.53 l/h/kg) in rats was about 1000-fold slower than the in vitro intrinsic clearance (606 l/h/kg) estimated by measuring the formation of U-97924 in rat liver microsomal incubations. Since U-89843 is extensively associated with serum proteins a poor extraction ratio into the liver may account for the slower biotransformation of U-89843 in vivo as compared to that exhibited in in vitro serum-free hepatocyte incubations. Addition of bovine serum albumin (1-40 mg/ml) to the in vitro UDS assay medium decreased the UDS mean net grains per nucleus response of U-89843. These results suggest that the effect of serum protein should be considered when comparing serum-free in vitro UDS and in vivo UDS results for highly serum protein bound compounds.
Zhao
Z
Z
Pharmacia and Upjohn Inc., Kalamazoo, MI 49001, USA. zhiyang.zhao@am.pnu.com
Koeplinger
K A
KA
Padbury
G E
GE
Aaron
C S
CS
Harbach
P R
PR
Mayo
J K
JK
Mattes
W B
WB
Bundy
G L
GL
D000818:Animals; D001798:Blood Proteins / Q000187:drug effects / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D008099:Liver / Q000187:drug effects*; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D009152:Mutagenicity Tests / Q000379:methods*; D011743:Pyrimidines / Q000378:metabolism / Q000633:toxicity*; D011758:Pyrroles / Q000378:metabolism / Q000633:toxicity*; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D012709:Serum Albumin / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism / Q000494:pharmacology
D001798:Blood Proteins; D011743:Pyrimidines; D011758:Pyrroles; D012709:Serum Albumin; C097295:U 89843; D012710:Serum Albumin, Bovine; D004247:DNA